Biologisten makromolekyylien kolmiulotteisten rakenteiden m ritt minen liuostilassa
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Biologisten makromolekyylien kolmiulotteisten rakenteiden määrittäminen liuostilassa. Nobel. 2002. Kurt Wüthrich. Proteiinien NMR-spektroskopia. Primaarirakenteen määrittäminen Sekundaari ja tertiäärirakenteen tai konformaation määrittäminen Kinetiikan ja molekulaarisen liikkeen tutkimus

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Biologisten makromolekyylien kolmiulotteisten rakenteiden määrittäminen liuostilassa

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Biologisten makromolekyylien kolmiulotteisten rakenteiden määrittäminen liuostilassa

Nobel

2002

Kurt Wüthrich


Proteiinien NMR-spektroskopia

  • Primaarirakenteen määrittäminen

  • Sekundaari ja tertiäärirakenteen tai konformaation määrittäminen

  • Kinetiikan ja molekulaarisen liikkeen tutkimus

  • Molekulaaristen vuorovaikutusten tutkimus


?

Rakenteen määrittämisenidea


Proteiinin NMR-spektri1957

Nobel

E. Purcell

1952

Felix Bloch

bovine pancreatic ribonuclease

Saunders, Wishnia and Kirkwood


NMR-spektroskopian perusajatus

Jokainen atomi, jolla on magneettinen ydin,

antaa yksittäisen signaalin,

joka sisältää informaatiota

paikallisesta kemiallisesta ympäristöstä,

rakenteesta ja dynamiikasta.


Magneettinen ydin

Jokainen atomi, jolla on magneettinen ydin ....

Stabiileja isotooppeja

1H, ~100%

13C, ~1.1%

15N, ~0.4%

31P, ~100%

2H, ~ 0%

Pieni magneettinen momentti

~ sauvamagneetti


Miten valmistan NMR-näytteen?

1H, 31P-leimaus sellaisenaan

  • 13C, 15N-leimaus

  • Proteiinit: tuotetaan rikastetuista lähtöaineista:

  • 13C-glukoosi, 15N-ammonium suolat

  • DNA: PCR (lyhyet ketjut)

  • RNA: In vitro synteesi

  • 2H-leimausproteiini tuotetaan 2H-rikastetussa

  • mediumissa


Proteiinin NMR-spektri

... yksittäinen signaali ...


Paikallinen kemiallinen (~magneettinen) ympäristö

  • NMR-spektrometrin kenttä

  • paikallinen kenttä

  • -lähellä olevat ytimet

  • -ympäröivät elektronipilvet


Spektroskopian perusteista

Energia kaavio – Purcellin kuva

B/E


Resonanssispektroskopia

B

aika

Fourier

muunnos

Vektorimalli – Blochin kuva

taajuus


f1

t1

t2

f2

Kaksiulotteinen korrelaatiospektroskopia

Signaalin modulaatio ajan t1 kuluessa

S(t1, t2)

S(f1, f2)

 FT 

Aikaulottuvuus  Fourier muunnos  taajuusulottuvuus (spektri)


NMR-spektroskopia kahdessa ulottuvuudessa

1991

Richard Ernst

Jean Jeener


NMR of Biological MacromoleculesMultidimensional Multinuclear Spectroscopy

Structural Biology


How to Interpret Spectra?

?

  • Structural implications

  • Atom type (and near neighbours)

  • Spatially near neighbours

  • Chemically bonded neighbours

  • Dynamic consequences

  • Fluctuating magnetic environment

  • Spectral parameters

  • Resonance frequency

  • Modulation of frequency

  • Correlation via dipolar field

  • Correlation indirectly via electrons (scalar coupling)

  • Relaxation


Magnetic EnvironmentDispersion of resonances

  • External magnetic field of the NMR-spectrometer

  • Local fields due to

  • -adjacent nuclei

  • -surrounding electron clouds

  • Chemical shift

  • = g(1 - s)B

    s is shielding (tensor)


Assignment of Resonances

Proteins display large dispersion

because they contain distinct

magnetic microenvironments.


Assignment of ResonancesIdentification of Residues by Characteristic Chemical Shifts

Aliphatic carbon shifts are particularly

characteristic for the residues.


Assignment of Backbone ResonancesPrinciple – Sequential Walk

HNCA

H R H R H R

| | | | | |

-N–Ca– C –N– Ca– C –N– Ca– C-

| || | || | ||

H O H O H O

Ca

S( Hi, Ni, Cai, Cai-1 )

N

H

HN(CO)CA

H R H R H R

| | | | | |

-N–Ca– C –N– Ca– C –N– Ca– C-

| || | || | ||

H O H O H O

Ca

S( Hi, Ni, Cai-1 )

N

H


HNCA

HN(CO)CA


Assignment of ResonancesSequential Walk via HNCO and HN(CA)CO


The redundancy in many alternatives for sequential assignment

is important for automated assignment.


Spectra Contain Implict Structural DataNOEs  Short Distances

NOEs

Nuclear Overhauser

Enhancement

i.e. dipole-dipole

relaxation.


Short Range Distances (NOEs)

ri = rref(Sref/Si)1/6


Spectra Contain Implict Structural DataScalar Couplings  Dihedrals

Karplus curve


How to Convert Spectral Parameters to Explicit Structural Data?

  • Short (<5-7Å) distances

  • via nuclear Overhauser spectroscopy (NOE)

  • Torsion angles

  • via scalar couplings (J-couplings)

  • Angles

  • via residual dipolar couplings (RDC)

  • Hydrogen bonds

  • via correlation spectroscopy

  • Secondary structures

  • via chemical shifts (resonance frequences)


T

t

Computation of Structure

Conversion of structural data to restraints

expressed as pseudo potentials

Restrained molecular dynamics (MD)

(Cartesian or torsion angle)


Result – Family of Structures

All structures that satisfy restraints

(within experimental error) are possible.


Evaluation of Structure

  • Accuracy

  • Restraint violations

  • Inconsitancies

  • Ramachandran violations

  • Precision

  • Spread of the family

  • Number of restraints

  • per residue


Direct Inspection of Spectra

Observing binding

Mapping binding epitopes

Detecting

conformational changes


About Field Fluctuations

”Reasons”

”Spectral Manifestations”

  • Bond vibrations

  • from pico to nano seconds

  • Conformational changes

  • from micro to milli seconds

  • Chemical exchange

  • from micro seconds to days

  • Relaxation measurements

  • -> rate constants, order

  • parameters, correlation times

  • Relaxation measurements

  • -> dispersion of parameters

  • Line width analysis

  • -> rate constants

Motional model


Conformational Exchange


Conformational Exchange


Conformational Exchange


Relaxation Dispersion

Transverse relaxation rates

vs. effective field and temperature

Frans A.A. Mulder et al.Nature Structural Biology 8, 932 - 935 (2001)


Hydrogen Exchange

Monitoring signal intensity

after dissolving to D2O

Denis Canet et al.Nature Structural Biology - Published online: 11 March 2002,


Hydrogen Exchange

Denis Canet et al.Nature Structural Biology - Published online: 11 March 2002,


Reaction Dynamics

Elan Zohar Eisenmesser,1 Daryl A. Bosco,1 Mikael Akke,2 Dorothee Kern1*

Science - Feb 2002,


Reaction DynamicsHot Spots


Reaction DynamicsProlyl Cis-Trans Isomerase


Dilute Liquid CrystalAnisotropic Medium


Distribution of Molecular Orientations


Vfree

Net Alignment

Vrestricted

D = Dmax(3cos2q-1)/2


N

A

D

C

B

Residual Dipolar Couplings


Alignments of Conformations


Monitoring Birth of Structure


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